The use of aerosolized Yersinia pestis as a biological weapon could potentially produce massive numbers of casualties. Weaponized plague could be rendered resistant to most antibiotics and delivered via an aerosol. Thus, in order to protect the military and civilian populations from both endemic and recombinant Y. pestis, new biological countermeasures must be developed that target essential pathogen vulnerabilities. The Y. pestis type III secretion (TTS) system enables the bacterium to subvert or destroy eukaryotic cells via the delivery of anti-host effector proteins. Disruption of the TTS process renders Y. pestis avirulent, indicating that the TTS process represents an attractive target for therapeutic intervention. Assembly of the TTS apparatus requires the participation of at least 21 Ysc (Yersinis secretion) proteins, Ysc proteins are hypothesized to assemble into a supramolecular structure that functions as a protein secretion and injection device. We propose to investigate the assembly and structure of the TTS apparatus. In Specific Aim 1, experiments are described to identify the protein interactions involved in the assembly of the TTS complex. Immunoprecipitation methodologies and yeast two- and three- hybrid studies will identify Ysc proteins that directly interact with one another. The overall objective of Specific Aim 2 is to establish conditions for the solubilization, purification and visualization of the assembled or partially assembled TTS complex. Environmental scanning electron microscopy (ESEM) of fixed samples and/or transmission electron microscopy (TEM) of negatively-stained samples will be used to visualize the surface-exposed and/or isolated TTS complex and/or partially assembled intermediates of this complex. In Specific Aim 3, experiments designed to identify and characterize chromosomally-encoded proteins that are required for the assembly and function of the TTS complex are described. Completion of these Specific Aims will not only provide essential information about the assembly and function of the Y. pestis TTS complex, but may also facilitate the development and/or design of novel therapeutics that target this essential virulence system.